Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production

Abstract

Background: Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL).

Methods: In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors.

Results: We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells.

Conclusions: Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria.

Clinical trials: Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007 . Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540 . Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027 . Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145 . Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283 . Registered May 18, 2012.

Keywords: Adipose-derived mesenchymal stromal cells; CD markers; Flow cytometry; Human platelet lysate; Manufacturing; RNA-sequencing; Release criteria.

Figures

Fig. 1

Traditional phenotyping of clinical-grade adipose-derived mesenchymal stromal cells (AMSCs) expanded in human platelet lysate. a Clinical-grade AMSCs grown in human platelet lysate were expanded ex vivo and immunophenotyped using flow cytometry according to the release criteria presented in this table. b Representative flow cytometry scatter plots show AMSCs are a homogeneous population of cells and exhibit surface expression of standard cell surface markers, including CD105, CD44, CD73, and CD90, and are negative for HLA-DR. c Analysis of the flow cytometry release criteria across clinical-grade AMSCs from 15 donors demonstrated minimal variability in the population frequency (% Gated) of the surface markers. All AMSC donor cells were >85 % positive for CD90, CD105, CD73, CD44, and HLA-ABC, and were <85 % positive for HLA-DR, CD45, and CD14

Fig. 2

Gene expression profiling and validation of cell surface markers across multiple mesenchymal cell types. a Gene expression of traditional markers by adipose-derived mesenchymal stromal cells (AMSCs) was analyzed using quantitative PCR (qPCR). AMSCs have relatively high expression levels of CD44, CD90, CD105, and CD73, and low or no expression of CD14 and CD45. To identify AMSC specific surface markers, high-throughput qPCR screening of 69 surface markers curated from the literature was performed on various mesenchymal cell types, including AMSCs (n = 4), bone marrow-derived stromal cells (BMSCs) (n = 2), primary bone cells (b) (n = 4), primary chondrocytes (C) (n = 4), and primary fibroblasts (F) (n = 4). b Hierarchical clustering analysis of qPCR data across multiple cell types shows that AMSCs have a unique phenotype at the gene expression level. c Comparison of different cell types revealed that classical surface markers, including CD44, CD73, and CD90, are expressed by not only AMSCs but also other cell types. Furthermore, nine non-classical markers were selected based on differential expression between the various mesenchymal cells. These markers, together with classical markers, were used to develop a novel antibody panel to characterize AMSCs

Fig. 3

Validation of nine non-classical markers by flow cytometry among 15 clinical-grade adipose-derived mesenchymal stromal cells (AMSCs). Expression of five classical (a) and nine non-classical markers (b) was validated by flow cytometry across 15 additional freshly isolated and expanded AMSC donors. Surface marker expression was evaluated by the percentage of the gated cell population and mean fluorescence intensity (MFI). c Representative histograms of surface marker expression compared to unstained AMSCs (negative control). Of the non-classical markers, CD36 exhibited two cell populations which varied from patient-to-patient

Fig. 4

Expression of novel markers by quantitative PCR (qPCR) and flow cytometry. Gene expression data were compared to flow cytometry data for two donors [adipose-derived mesenchymal cell (AMSC) donors 1 and 4]. Highly abundant markers showed good concordance (top panel) between the techniques, whereas lower abundance markers showed variability (bottom panel). In particular, CD200 and CD274 were not correlated. MFI mean fluorescence intensity

Fig. 5

Effect of cryopreservation on surface marker expression. Flow cytometry for all 14 markers was performed on samples from 5 adipose-derived mesenchymal stromal cell (AMSC) donors before cryopreservation (pre-freeze), immediately after rescue from cryopreservation (post-thaw), and 4 days after revival from cryopreservation (4d Culture). a AMSCs were >90 % positive for classical surface markers across all manufacturing conditions, except CD34 which was a negative marker. b Non-classical surface markers exhibited variability both in population positivity (%Gated) and mean fluorescence intensity (MFI) as cells were processed through the various manufacturing conditions. One-way ANOVA and post-hoc testing were performed to identify variables that were statistically significant at p < 0.05

Fig. 6

High-resolution RNA-sequencing (RNA-seq) analysis of surface marker gene expression by proliferating and confluent adipose-derived mesenchymal stromal cells (AMSCs). a Gene expression profiling using quantitative PCR for 69 cell surface protein-encoding genes reveals some surface markers are differentially expressed between proliferating (~70–80 % confluent) and confluent (100 % confluent) AMSCs. Values indicate fold-change (Log10 transformed) of confluent over proliferating, and are averages of samples from four different AMSC donors. Fold-change analysis shows markers that are differentially expressed between proliferating and confluent cultures. To further evaluate differential surface marker expression, RNA-seq was performed on proliferating and confluent AMSCs from four different donors. b Expression values for 551 cell surface genes expressed at a magnitude >0 reads per kilobase per million (RPKM) by all AMSC donors were extracted from the RNA-seq data set and subjected to hierarchical clustering analysis, which revealed distinct expression patterns for proliferating and confluent cells. c Representative graphs of genes derived from RNA-seq analysis shows CD292/BMPR1A was constitutively expressed, CD168/HMMR was only expressed by proliferating cells, and CD106/VCAM1 was only expressed in confluent cells